The Ras gene is a protooncogene, first obtained by cloning from Harvey, Kirsten rat sarcoma, referred to as HRas and KRas[1]. Later, another similar gene was found when the human neuroblastoma DNA infected the NIH3T3 cell, and it was called NRas, and the three are the most important members of the Ras gene family[2]. In function, the Ras protein plays the role as a molecular switch, and can regulate the growth of cells under normal expression. Abnormal conditions such as point mutation, overexpression or gene translocation can lead to abnormal proliferation of cells, and finally result in the formation of tumor. Ras mutation exists in over 30% of human tumors[3, 4]. Presently, the most deeply researched is KRas, and its overexpression and mutation generally occur in many cancers such as thyroid cancer, breast cancer, and among the North American pulmonary granule cancer patients, the mutation rate is as high as 25%. Also, KRas is related to TKI resistance, therefore it has become the molecular mark in the diagnosis and treatment of many tumors, playing an important clinical role[5, 6]. As another member of the Ras family, NRas has many similarities to KRas in both structure and function, and with the deepening of researches in recent years, it has gradually become another molecular indicator serves as an important basis for clinical disease evaluation and treatment besides the KRas.
The NRas gene locates on the short arm of human Chromosome 1 (1p22-p32), encodes p21 protein having 189 amino acids[7]. The NRas proteins has a homology as high as 85% with other proteins in the Ras family, and these highly conservative structure domains include those playing an extremely important role in the function of protein, such as the binding domain of the guanosine triphosphate (GTP) and the effector molecules, and the CAAX sequence motif that positions the NRas protein on the farnesyl transferase acting site—C end on the plasmalemma[8, 9]. Therefore, functionally, NRas also has many common features of the RAS family proteins: it locates on the inside of the cell membrane, and is a G protein of low molecular weight, with very strong affinity to guanylic acid, and having GTPase activity; it has two conformations of GTP binding (Ras.GTP) and GDP binding (Ras.GDP), and interconversion can take place of the two under certain conditions; the Ras protein is in the inactivated state when binds with GDP, and in the activated state when binds with GTP, such that it activates the downstream signal pathway, so it plays an extremely important switching role in the signal transduction[10, 11]. The downstream signal pathway activated by NRas is now the RAS signal pathway having been most clearly researched currently: the PTK (protein tyrosine kinase)-Grb2 (growth factor receptor-bound protein2)-Ras-Raf-MAPK (mitogen activated protein kinase)-ERK (extracellular signal-regulated kinase) pathway[12]. When exogenous stimulus such as growth factor (EGF) is bound with cell membrane receptor (EGFR), so that the corresponding tyrosine kinase on the receptor is phosphorylated, the phosphorylated tyrosine residue, after binding with the SH2 zone of Grb2, recruits the ornithine exchange factor (SOS) to bind with the SH3 zone of Grb2, to form the complex Grb2-SOS, and this complex binds with Ras and converts Ras-GDP into Ras-GTP, thus activating the Ras. The activated Ras then activates the downstream Raf kinase, the Raf kinase phosphorylates MAPK, the MAPK activates the ERK. After the ERK is activated, it transfers to inside the cell nucleus, directly activating various transcription factors of c-myc, so as to participate in various physiological processes such as cell growth, development, division and differentiation[13]. Mutation of NRas can lead to abnormal activation of the downstream Raf and MAPK etc., thus playing an important role in the tumor malignance[14].
Mutation of Ras protein mainly occurs on Codons 12, 13, 59 and 61, and the mutation probability is the highest on Codons 12 and 61[15]. The mutation is of different types in different cancers. In non-small cell lung cancer, the main mutation is the guanine in Codon 12 is replaced by thymine, while in colon cancer, at the same position, mainly the guanine is replaced by adenine[16]. NRas mutation mainly occurs at Codon 61, with high probability in melanoma. Thoams et al used the “mutation” and “melanoma” as key words to retrieve and analyze articles published in the pubmed during 1966-2006, and found a NRas mutation rate as high as 28% in surface diffused and nodositas melanoma[17]. Vikas et al found 10 cases of NRas mutation (17%, 10/16) in 60 cases of primary melanoma, and all mutations occur at Codon 61[18]. It is worth our high attention that in recent years, NRas has been confirmed as the driver gene of lung cancer. Kris et al published on ASCO in 2011 a research from LCMC (NCI's Lung Cancer Mutation Consortium), in which the 10 driver genes including KRas, EGFR and NRas etc. in 1000 pulmonary granule cancer tissue specimens were tested. All patients were of phases IIIb/IV, with sufficient tissue specimens. The research included 830 patients, 60% patients had driver gene mutation, and the mutation rate of NRas was 0.2%[19, 20].
As the effect of Ras mutation on tumors is mostly concentrated on the Ras/Raf/MEK/ERK pathway, therefore today, the targeted antitumor drugs against Ras mutation are concentrated on the targeted intervention at different nodes in this signal pathway. The developed drugs include the farnesyl transferase inhibitors (FTIs) that intervene with the Ras membrane binding and lypolyzation, such as tipifarnib[21], ATP indirect competitor sorafenib for activation of Raf[22, 23], and CI-1040 and AZD6244 for MEK targeted intervention, and the latter have already been used in clinical test[24]. As ERK is the only substrate of MEK known today, and it is usually described as the single pathway downstream the Ras, therefore it is generally believed that inhibition solely at the MEK target point is equal to blocking the ERK activation caused by the mutated Ras, and it can also avoid the effect of invalid targeting caused by the interference from other pathways[9].
It should be particularly pointed out that, the latest researches have indicated that the NRas mutation is related to the TKI resistance in the treatment of lung cancer. Compared with the gefitinib sensitive PC-9 cell (PC-9/WT), no EGFR-TKIs resistance gene such as KRas and HER2 were detected in PC-9 cell (PC-9/gef) where gefitinib resistance was produced, but NRas mutation at Codon 61 was found. Moreover, in the condition that an administration of gefitinib or AZD6244/CI1040 only could not make cell apoptosis, the combined use of both drugs can effectively promote cell apoptosis[25]. These experimental results have indicated that NRas mutation may play an important role in the TKI resistance in lung cancer treatment, and this has provided new basis and possibility for the detection and treatment of lung cancer.
In general, more and more evidences have shown that NRas mutation has important significance in the occurrence and development of many human tumors such as melanoma and lung cancer. The detection of its mutation enables accurately prediction of the effectiveness of the corresponding targeted drug treatment, so as to facilitate clinical selection of drugs, effectively improve treatment results and provide the maximum benefit to patients; in the meanwhile, it can also avoid medical expense burden on patients and waste of public medical resources resulted from unreasonable application of drugs, and reduce unnecessary loss of time and money losses.